Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required

The mechanisms by which insulin deficiency affects muscle glucose transport were investigated. Epitrochlearis muscles from rats with streptozotocin-induced diabetes and from controls were incubated in vitro for 0.5-14 h. The incubation was shown not to impair muscle energy stores or tissue oxygenation. Diabetes decreased basal 3-O-methylglucose transport by 40% (p less than 0.01), and insulin-stimulated (20 milli-units/ml) glucose transport capacity by 70% (p less than 0.001). In vitro incubation gradually normalized insulin responsiveness (3.77 +/- 0.38 before versus 8.97 +/- 0.65 mumol X ml-1 X h-1 after 12 h of incubation). Basal glucose transport remained significantly reduced. The reversal of the insulin responsiveness did not require the presence of rat serum and, furthermore, took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 microunits/ml insulin (9.85 +/- 0.59 versus 8.06 +/- 0.59 mumol X ml-1 X h-1, p less than 0.05). Glucose at 30 mM did not affect the normalization of the insulin-stimulated glucose transport capacity, whereas incubation in serum from diabetic rats resulted in a slightly (26%) blunted reversal (7.60 +/- 0.39 versus 8.89 +/- 0.45 mumol X ml-1 X h-1 with diabetic versus control serum for 14 h, p less than 0.05; before incubation the value was 3.87 +/- 0.40). Inhibition of protein synthesis by cycloheximide blocked the normalization by 80%. These results suggest the presence in diabetic serum of some labile factor that might inhibit the glucose transport system. The results indicate that the decreased insulin-stimulated glucose transport capacity, in the insulin-deficient diabetic muscle, is not a direct consequence of the lack of insulin or of high glucose concentrations.     

Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis.

We investigated the requirement for cellular cytoskeleton in CR- and FcR-mediated phagocytosis by human monocyte-derived macrophages (M phi). Inhibition of actin microfilament (MF) assembly and stability by cytochalasins B and D completely inhibited M phi phagocytosis of sheep E coated with C3b (EC3b), iC3b (EC3bi), and IgG (EIgG) via CR1, CR3, and FcR, respectively. Ligand-binding to either CR or FcR was not effected by cytochalasins. Nocodazole (NOC), which prevents microtubule (MT) polymerization, and taxol, which causes random polymerization of MT inhibited M phi phagocytosis of EC3b(i) but not EIgG. However, the combination of taxol (5 x 10(-4) M) and NOC (2 x 10(-6) M) augmented M phi CR-mediated phagocytosis. In addition, agents known to increase intracellular cGMP augmented phagocytosis of EC3b(i). Conversely, agents that increase intracellular cAMP inhibited CR-mediated phagocytosis. These agents had no effect on FcR-mediated phagocytosis, and did not effect ligand-binding to CR or FcR. PMA markedly enhanced CR- but not FcR-mediated phagocytosis, and augmentation of CR-mediated phagocytosis by PMA was inhibited by both CD and NOC. In contrast, the synthetic diacylglycerol, 1-oleoyl-2-acetoyl-sn-3-glycerol augmented, and inhibitors of protein kinase C inhibited M phi phagocytosis via CR and FcR. These data indicate that for adherently cultured human M phi: 1) binding of ligand-coated E to CR or FcR does not require an intact cytoskeleton; 2) intact actin microfilament are required for phagocytosis via CR and FcR; 3) phagocytosis via CR1 and CR3 but not FcR is dependent on MT assembly; 4) PMA most likely augments CR-mediated phagocytosis through promotion of MT assembly; and 5) PKC activity is involved in the phagocytic signal generated by both CR and FcR.     

Polymorphonuclear neutrophil dysfunctions in streptozotocin-induced type 1 diabetic rats

Since conflicting results have been reported on non-specific immune response in type 1 diabetes, this study evaluates polymorphonuclear neutrophil (PMN) functions in the infection free Long Evan diabetic rats (type 1) by using tests that include: polarization assay, phagocytosis of baker\'s yeasts (Saccharomyces cerevisiae) and nitroblue tetrazolium (NBT) dye reduction. Polarization assay showed that neutrophils from diabetic rats were significantly activated at the basal level compared to those from the controls (p < 0.001). After PMN activation with N-formylmethionyl-leucyl-phenylalanine (FMLP), control neutrophils were found to be more polarized than those of the diabetic neutrophils and the highest proportions of polarization were found to be 67 % and 57 % at 10(-7) M FMLP, respectively. In the resting state, neutrophils from the diabetic rats reduced significantly more NBT dye than that of the controls (p < 0.001). The percentages of phagocytosis of opsonized yeast cells by the neutrophils from control and diabetic rats were 87 % and 61 %, respectively and the difference was statistically significant (p < 0.001). Evaluation of the phagocytic efficiency of PMNs revealed that control neutrophils could phagocytose 381 +/- 17 whereas those from the diabetic rats phagocytosed 282 +/- 16 yeast cells, and the efficiency of phagocytosis varied significantly (p < 0.001). Further, both the percentages of phagocytosis and the efficiency of phagocytosis by the diabetic neutrophils were inversely related with the levels of their corresponding plasma glucose (p = 0.02; r = -0.498 and p < 0.05; r = -0.43, respectively), which indicated that increased plasma glucose reduced the phagocytic ability of neutrophils. Such relationship was not observed with the control neutrophils. These data clearly indicate that PMN functions are altered in the streptozotocin (STZ)-induced diabetic rats, and hyperglycemia may be the cause for the impairment of their functions leading to many infectious episodes.     

Delayed removal of 125I-labeled very low density lipoprotein from plasma of rats with insulin-deficient diabetes mellitus.

The present studies demonstrate that the removal rate of exogenously labelled 125I-VLDL-protein is prolonged when total serum from insulin-deficient rats combined with isolated 125I-VLDL is injected into normal recipient rats (6.8 +/- 0.7 vs 4.2 +/- 0.4 min; p < 0.01), but not when 125I-VLDL-protein is isolated and injected alone (4.2 +/- 0.8 vs 4.3 +/- 0.8 min). Furthermore, the present studies demonstrate that when isolated 125I-VLDL-protein is recombined with either VLDL-free (d > 1.006 g/ml), or lipoprotein-free serum (d > 1.215 g/ml) from insulin-deficient rats, the defect in removal rate of VLDL-protein observed in total serum is reestablished (125I-VLDL + VLDL-free serum from insulin-deficient rat vs that from normal rat: 7.6 +/- 1.2 vs 4.6 +/- 0.7 min, p < 0.05; and 125I-VLDL + lipoprotein-free serum from insulin-deficient rat vs that from normal rat: 6.4 +/- 0.7 vs 4.1 +/- 0.4 min, p < 0.01). These data suggest that a factor or factors exist in lipoprotein-free serum of insulin-deficient rats which interfere with the normal removal of 125I-VLDL. Since we have previously demonstrated a prolongation in the removal rate of endogenously labeled VLDL-3H-TG, the defect in removal of VLDL from the plasma of insulin-deficient rats appears to include both the lipid and protein moieties of the VLDL particles.     

Phospholipase A2IVα regulates phagocytosis independent of its enzymatic activity

Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.     

Insulin Binding to the Blood-Brain Barrier in the Streptozotocin Diabetic Rat

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Why does experimental insulin deficiency lead to a decrease in removal of very low density-triglyceride from plasma?

We have previously suggested that mechanisms other than reduced lipoprotein lipase (LPL) activity might contribute to the defect in plasma removal of very low density lipoprotein (VLDL)-triglyceride (TG) observed in insulin-deficient rats. To further evaluate this phenomenon, removal rates of TG in nonfractionated plasma, as well as in isolated lipoprotein fractions obtained from insulin-deficient and control rats, were compared in a new, sensitive in vivo bioassay system (estradiol-treated male rats with a consistently low endogenous VLDL-TG pool). Removal of TG in nonfractionated plasma from insulin-deficient rats was slower than that of control rats: 3.0 +/- 0.3 vs 1.6 +/- 0.2 min (P less than 0.001). No difference was found in removal rate of isolated VLDL-TG (2.5 +/- 0.3 vs 2.6 +/- 0.4 min), or in removal rates of TG carried in other lipoprotein fractions. We next determined the effect of injection into normal rats of aliquots of dialyzed lipoprotein-free (D greater than 1.215) plasma from insulin-deficient and control rats on the removal rate of normal VLDL-TG, and found that lipoprotein-free plasma from insulin-deficient rats significantly (P less than 0.01) prolonged removal of normal VLDL-TG (4.3 +/- 0.4 to 6.8 +/- 0.7 min). This same fraction did not interfere with the in vitro hydrolysis of normal VLDL-TG by post-heparin LPL. Thus, a factor in the D greater than 1.215 plasma fraction of insulin-deficient rats is present which interferes with the rate of removal of TG from plasma, unrelated to inhibition of LPL activity.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Low phospholipid arachidonic acid values in diabetic platelets.

Platelet aggregation is enhanced in diabetes mellitus, and platelets may be implicated in the pathogenesis of diabetic angiopathy. Increased platelet aggregation is probably mediated by the production of the proaggregatory prostaglandin thromboxane, which is synthesised from arachidonic acid (C20:4) by the action of the platelet enzymes cyclo-oxygenase and thromboxane synthetase. The fatty acid composition of platelet phospholipid was measured in 20 normal controls, 10 insulin-treated diabetics with no or minimal retinopathy, and 10 insulin-treated diabetics with proliferative retinopathy. The percentage of arachidonic acid was significantly higher in controls (mean 22.6%) than in the diabetics with no or minimal retinopathy (mean 18.5%; p less than 0.025) and the diabetics with proliferative retinopathy (mean 14.6%; p less than 0.001). The percentage of linoleic acid was lower in controls (mean 8.9%) than in the diabetics with no or minimal retinopathy (mean 12.6%; p less than 0.01) and diabetics with proliferative retinopathy (mean 13.1%; p less than 0.001). The mean percentage of linolenic acid was significantly lower in the diabetics with proliferative retinopathy (2.7%) than in the normal control group (4.4%; p less than 0.01). A significant negative correlation was found between the percentages of arachidonic acid and glycosylated haemoglobin (Rs = -0.58; p less than 0.001). A significant positive correlation was found between linoleic acid and the percentage of glycosylated haemoglobin (Rs = 0.51; p less than 0.01). The reciprocal correlation between percentages of arachidonic acid and glycosylated haemoglobin suggests that diabetic control may influence thromboxane release and platelet activity directly and that low percentages of arachidonic acid reflect the increased degree of in-vivo activation.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.     

Effect of streptozotocin-induced diabetes on the activity of 7 alpha-hydroxy-4-cholesten-3-one-specific 12 alpha-hydroxylase in rats

The activity of 12 alpha-hydroxylase in hepatic microsomes from normal, streptozotocin-induced diabetic, and insulin-treated diabetic rats was studied with 7 alpha-hydroxy-4-cholesten-3-one as a substrate. In the diabetic rats, the 12 alpha-hydroxylase activity was about 50% lower than in the normal rats. Treatment of the diabetics with insulin cancelled the reduction of the activity. These results show that an insulin-deficient state causes a paradoxical decrease in the activity of the key enzyme for cholic acid formation.     

Morphological and hormonal changes following vasectomy in rats, suggesting a functional role for Leydig-cell associated macrophages.

The effects of bilateral vasectomy on hormone serum levels as well as Leydig cell and associated macrophage structure were analysed in parallel in rats 36 weeks following the operation. Serum testosterone was decreased in vasectomized rats (1.96 +/- 0.11 ng/ml) compared with control animals (3.44 +/- 0.22 ng/ml, p less than 0.05). Vasectomy also resulted in an increase in serum luteinizing hormone (LH) to 0.299 +/- 0.02 ng/ml compared to the control group (0.175 +/- 0.01 ng/ml, p less than 0.05). Also serum follicle-stimulating hormone (FSH) was increased following vasectomy (350.88 +/- 15.5 ng/ml) compared to 132.0 +/- 4.8 ng/ml in control animals (p less than 0.01). Morphometric analysis of Leydig cells showed hypertrophy with a 19% increase of total cell area, p less than 0.01 (cytoplasm 28%, nucleus 8% increase). On the ultrastructural level, leydig cells demonstrated massively dilated smooth endoplasmic reticulum characteristic for stimulated cells. There was also a significant hypertrophy of the Leydig cell-associated macrophages. The macrophage cell area was enlarged by 22%, p less than 0.01 (cytoplasm 25%, nucleus 18%). Vasectomy also led to remarkable ultrastructural changes of macrophages with a marked dilated and extended rough endoplasmic reticulum. Macrophages were found in apposition to Leydig cells with close cellular contact zones, and they frequently formed cell extensions on Leydig cells. Our data obtained following vasectomy indicate that, by their close contacts to Leydig cells, as well as the known influence on Leydig-cell steroidogenesis, macrophages may form the basis of a local immunoendocrine regulation of the pituitary-gonadal axis.     

Decreased Fc receptor avidity and degradative function of monocytes from patients with systemic lupus erythematosus

We studied the binding and degradation of stable, soluble heat aggregates of 125I-IgG (A-IgG) by monocytes from 30 patients with systemic lupus erythematosus (SLE) and 30 normals. Relative avidities (KE) for Fc receptor (FcR) binding of A-IgG and maximal binding of A-IgG by monocytes were determined from Scatchard plots of binding data obtained at 4 degrees C. Rates of degradation (Vmax) of A-IgG at 37 degrees C were calculated from Lineweaver-Burke plots of the Michaelis-Menton equation. KE were decreased in SLE monocytes (15.5 X 10(-9) L/M) as compared with normals (20.1 X 10(-9) L/M, p less than 0.005) and Vmax were decreased for SLE (0.89 ng/hr) as compared with normals (1.11 ng/hr, p less than 0.005). The maximal FcR binding by SLE monocytes was not statistically different in SLE patients and normals, but monocytes from SLE patients with active disease showed a lower maximal binding capacity for A-IgG (4.9 ng/10(5) cells) than normals (5.4 ng/10(5) cells, p less than 0.05). KE and Vmax in SLE were also lower for patients with active disease than for normal subjects. KE in patients whose anti-ssDNA binding was greater than 20% were lower than for those with DNA binding of less than 20% (p less than 0.005). These data suggest that patients with active SLE have diminished numbers of available FcR on their circulating monocytes, possibly due to interiorization of FcR during endocytosis of endogenous circulating immune complexes.     

TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonised particles in macrophages

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.     

ANG II enhances contractile responses via PI3-kinase p110 delta pathway in aortas from diabetic rats with systemic hyperinsulinemia

We investigated the involvement of ANG II and phosphatidylinositol 3-kinase (PI3-K) in the enhanced aortic contractile responses induced by hyperinsulinemia in chronic insulin-treated Type 1 diabetic rats. Plasma ANG II levels were elevated in untreated compared with control diabetic rats and further increased in insulin-treated diabetic rats. Aortic contractile responses and systolic blood pressure were significantly enhanced in chronic insulin-treated diabetic rats compared with the other groups. These insulin-induced increases were largely prevented by cotreatment with losartan (an ANG II type 1 receptor antagonist) or enalapril (an angiotensin-converting enzyme inhibitor). LY-294002 (a PI3-K inhibitor) diminished the increases in contractile responses in ANG II-incubated aortas and aortas from chronic insulin-treated diabetic rats. The norepinephrine (NE)-stimulated levels of p110 delta-associated PI3-K activity and p110 delta protein expression were increased in aortas from insulin-treated diabetic compared with control and untreated diabetic rats, and chronic administration of losartan blunted these increases. Contractions were significantly larger in aortas from diabetic rats incubated with a low concentration (inducing approximately 10% of the maximum contraction) of ANG II or with NE or isotonic K+ than in aortas from nonincubated diabetic rats. NE-stimulated p110 PI3-K activity was elevated in aortas from diabetic rats coincubated with a noncontractile dose of ANG II. These results suggest that, in insulin-treated Type 1 diabetic rats with hyperinsulinemia, chronic ANG II type 1 receptor blockade blunts the increases in vascular contractility and blood pressure via a decrease in p110 delta-associated PI3-K activity.     

Effects of inhibitors of C1q biosynthesis on macrophage Fc receptor subclass-mediated antibody-dependent cellular cytotoxicity and phagocytosis

We examined the effects of the inhibitors of C1q or collagen biosynthesis, 2,2'-dipyridyl (DP), and 3,4-dehydro-DL-proline (DHP) on murine macrophage (M phi) FcR subclass-mediated antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of sheep erythrocyte targets. Oil-elicited peritoneal M phi from C3HeB/FeJ mice which were cultured for 24 hr with DP (0.08 or 0.10 mM) or DHP (0.8 or 1.0 mM) showed a significant decrease in FcR subclass-mediated ADCC for murine monoclonal IgG2a (FcRI) and IgG2b/IgG1 (FcRII) as well as for heterologous polyclonal IgG. These collagen inhibitors also blocked phagocytosis mediated by both IgG2a- and IgG2b-opsonized erythrocytes. DP was more potent than DHP in blocking FcR effector functions in a reversible fashion and neither inhibitor affected M phi C3b receptor function. Pretreatment of M phi with collagenase resulted in significant reduction in FcR-mediated ADCC and phagocytosis. The inhibition of M phi FcR subclass-mediated ADCC and phagocytosis by collagen C1q synthetic inhibitors or by collagenase treatment further confirms a functional relationship between cell-associated C1q and FcR-dependent functions.     

Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle

The cellular composition of ovine corpora lutea obtained during the early (Day 4), mid (Days 8 and 12), and late (Day 16) stages of the estrous cycle was determined by morphometric analysis. Individual corpora lutea were collected via midventral laparotomy from a total of 19 ewes. A center slice from each corpus luteum was processed for electron microscopy and subsequent morphometric analysis of the numbers and sizes of steroidogenic and nonsteroidogenic cells. Luteal weight progressively increased throughout the estrous cycle (p less than 0.05). Corpora lutea collected on Day 16 were assigned to one of two subgroups on the basis of gross appearance and weight: nonregressed (NR, 542 +/- 25 mg) or regressed (R, 260 +/- 2 mg). There were no significant changes in the proportion of the corpus luteum occupied by small luteal cells (19 +/- 2%) or large luteal cells (36 +/- 1%) throughout the estrous cycle. The total number of steroidogenic cells per corpus luteum increased from 21.8 +/- 3.7 (X 10(6)) on Day 4 to 61.7 +/- 5.4 (X 10(6)) on Day 8 (p less than 0.05) and remained elevated thereafter. The number of small luteal cells was 10.0 +/- 2.7 (X 10(6)), 39.7 +/- 1.4 (X 10(6)), 46.1 +/- 5.8 (X 10(6)), 49.0 +/- 13.7 (X 10(6)), and 29.9 +/- 8.6 (X 10(6)) on Days 4, 8, 12, 16 (NR), and 16 (R), respectively (p less than 0.05, Day 4 vs. Days 8, 12, 16 NR). In contrast, the number of large luteal cells was 11.8 +/- 1.5 (X 10(6)) on Day 4 and did not vary significantly during the remainder of the estrous cycle. The numbers of nonsteroidogenic cell types increased (p less than 0.05) from Day 4 to Day 16 (NR) but were decreased in regressed corpora lutea (Day 16 R). Regression was characterized by a 50% decrease (p less than 0.05) in the total number of cells per corpus luteum from 243 +/- 57 ( X 10(6)) on Day 16 (NR) to 125 +/- 14 ( X 10(6)) on Day 16 (R) (p less than 0.05). Small luteal cells remained constant in volume throughout the entire estrous cycle (2520 +/- 270 microns 3), whereas large luteal cells increased in size from 5300 +/- 800 microns 3 on Day 4 to 16,900 +/- 3300 microns 3 on Day 16 (NR) (p less than 0.05). In summary, small luteal cells increased in number but not size throughout the estrous cycle, whereas large luteal cells increased in size but not number.     

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.     

Ganglioside treatment of diabetic rats; effects on nerve adenosine triphosphatase activity and motor nerve conduction velocity

Ouabain-sensitive ATPase activity (expressed as nmol ADP produced/h/mg (wet) nerve +/- SEM) was measured in homogenates of sciatic nerve from control rats and rats with streptozotocin-induced diabetes of 8 wk duration. Nerves from diabetic rats showed activity (21.7 +/- 2.0) which was significantly (p less than 0.05) less than that of controls (34.6 +/- 4.8). These animals also showed a deficit in conduction velocity (m/sec +/- SEM) of sciatic nerve motoneurones (50.7 +/- 0.4 vs. 57.7 +/- 0.7 in controls; p less than 0.001). In parallel, matched control and diabetic groups were treated daily with mixed gangliosides extracted from bovine brain (10 mg/kg i.p.). After such treatment for 8 wk the deficit in ouabain-sensitive ATPase activity did not develop in the diabetic group (treated diabetics, 31.9 +/- 3.7; treated controls, 34.5 +/- 3.8). However, the treatment did not affect the deficit in motor nerve conduction velocity (treated diabetics, 50.9 +/- 1.1 vs. treated controls, 57.9 +/- 0.5; p less than 0.001). Accumulations of the polyol pathway metabolites--sorbitol and fructose--together with depletion of nerve myo-inositol were similar in both diabetic groups. These data indicate an etiology for the conduction velocity deficit which differs from that of the deficit in ouabain-sensitive ATPase.     

Slow Axonal Transport of Structural Polypeptides in Rat, Early Changes in Streptozocin Diabetes, and Effect of Insulin Treatment

The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin-treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 +/- 0.1 mm/day (mean +/- SD). The actin-related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 +/- 0.2 mm/day. For alpha- and beta-tubulin we found an apparent transport velocity of 2.2 +/- 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 +/- 0.19% of trichloroacetic acid-insoluble radioactivity versus 0.69 +/- 0.17% in controls; 2p less than 0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament subunits. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic injury was observed.